CA2159830C

CA2159830C - System for real time detection of nucleic acid amplification products

Info

Publication number: CA2159830C
Application number: CA002159830A
Authority: CA
Inventors: Timothy M Woudenberg; Kevin S. Bodner; Charles R. Connell; Alan M. Ganz; Lincoln J. Mcbride; Paul G. Saviano; John Shigeura; David H. Tracy; Eugene F. Young; Linda G. Lee
Original assignee: PE Corp
Current assignee: Applied Biosystems LLC
Priority date: 1994-04-29
Filing date: 1995-04-19
Publication date: 2001-07-03
Anticipated expiration: 2015-04-19
Also published as: US6015674A; AU2389695A; WO1995030139A1; JP2909216B2; AU698953B2; EP0706649B1; CA2159830A1; JPH08510562A; ATE198511T1; DE69519783D1; US5928907A; DE69519783T2; EP0706649A1

Abstract

A system is provided for carrying out real time fluorescence-based measurements of nucleic acid amplification products. In a preferred embodime nt of the invention, an excitation beam is focused into a reaction mixture through a surface, the reaction mixture containing: (i) a first fluorescent indicator capable of generating a first fluorescent signal whose intensity i s proportional to the amount of an amplification product in the volume of the reaction mixture illuminated by the excitation beam and (ii) a second fluorescent indicator homogeneously distributed throughout the reaction mixture capable of generating a second fluorescent signal proportional to th e volume of reaction mixture illuminated by the excitation beam. Preferably, t he excitation beam is focused into the reaction mixture by a lens through a portion of a wall of a closed reaction chamber containing the reaction mixture. The same lens is used to collect the first and second fluorescent signals generated by the first and second fluorescent indicators, respectively, in response to the excitation beam. The ratio of the fluoresce nt intensities of the first and second fluorescent signals provides a stable quantitative indicator of the amount of amplification product synthesized in the course of the amplification reaction.

Description

_s ' ,.
2159830 _ SYSTEM FOR REAL T>ME DETECTION OF NUCLEIC ACID
AMPLIFICATION PRODUCTS
The invention relates generally to the field of nucleic acid amplification, and more particularly to a system for measuring in real time polynucleotide products from nucleic acid amplification processes, such as polymerase chain reaction (PCR).
Back, ound Nucleic acid sequence analysis is becoming increasingly important in mater research, medical, and industrial fields, e.g. Caskey, Science 236: 1223-1228 (1987);
Landegren et al, Stience, 242: 229-237 (1988); and Arnheim et al, Ann. Rev_ Biochem., 61: 131-156 (1992). The development of several nucleic acid amplification schemes has played a critical role in this trend, e.g. polymerase chain reaction (PCR), Tnnic et al, editors, PCR Protocols (Academic Press, New York, 1990); McPherson et al, editors, PCR: A Practical Approach (BtL Press, Oxford, 1991); ligation-based amplification techniques, Bara~, PCR Methods and Applications 1: 5-16 (1991); and the floe.
PCR in particular has become a research tool of major importance with applications is cloning, analysis of genetic expression, DNA sequencing, genetic mapping, drug discovery, and the like, e.g. Arnheim et al (cited above);
Gr'lfland et al, Proc. Natl. Acad. Sci., 87: 2725-2729 (1990); Bevan et al, PCR Methods and Applications, 1: 222-228 (1992); Green et al, PCR Methods and Applications, 1:

90 (1991); Blackwell et al, Science, 250: 1104-1110 (1990).
A wide variety of instrumentation has been developed for carrying out nucleic acid amplifications, particularly PCR, e.g. Johnson et al, U.S. patent 5,038,852 (computer-controlled thermal cycler); Wittwer et al, Nucleic Acids Research, 17:
4353-4357 (1989)(capillary tube PCR); Hallsby, U.S. patent 5,187,084 (air-based temperature control); Garner et al, Biotechniques, 14: 112-115 (1993)(high-throughput PCR in 864-well plates); Wilding et al, International applicarioa No.
-1_ PCT/LTS93/04039 (PCR in micro-machined structures); Schnipelsky et al, European patent application No. 90301061.9 (publ. No. 0381501 A2)(disposable, single use PCR device), and the like. Important design goals fundamental to PCR
instrument development have included fine temperature control, minimization of sample-to-sample variability in multi-sample thermal cycling, automation of pre- and post-PCR
processing steps, high speed cycling, minimization of sample volumes, real time measurement of amplification products, minimization of cross-contamination, or sample carryover, and the like. In particular, the design of instruments that permit PCR to be carried out in closed reaction chambers and monitored in real time is highly desirable. Closed reaction chambers are desirable for preventing cross-contamination, e.g. FTiguchi et al, Biotechnology, 10: 413-417 (1992) and 11: 1026-1030 (1993); and Holland et al, Pros. Nail. Acad. Sci., 88: 7276-7280 (1991). Clearly, the successful realization of such a design goal would be especially desirable in the analysis of diagnostic samples, where a high frequency of false positives and false negatives would severely reduce the value of the PCR-based procedure. Real time monitoring of a PCR permits far more accurate quantitation of starting target DNA
concentrations is multiple-target amplifications, as the relative values of close concentrations can be resolved by taking into account the history of the relative concentration values during the PCR Real time monitoring also permits the efficiency of the PCR to be evaluated, which can indicate whether PCR inhibitors are present in a sample.
Holland et al (cited above) and others have proposed fluorescence-based approaches to provide real time measurements of amplification products during a PCR
Such approaches have either employed intercalating dyes (such as et6idium bromide) to indicate the amourn of double stranded DNA present, or they have employed probes containing fluoresces-quencher pairs (the so-called "Tac-Man" approach) that are cleaved during amplification to release a fluorescent product whose concentration is proportional to the amount of double stranded DNA present.
Unfortunately, successfiil implementation of these approaches has been impeded because the required fluorescent measurements must be made against a very high fluorescent background. Thus, even minor sources of instrumental noise, such as the formation of condensation in the chamber during heating and cooling cycles, formation of bubbles in an optical path, particles or debris in solution, differences in sample volumes-and hence, differences in signal emission and absorbence, and the like, have hampered the reliable measurement of the fluorescent signals.
In view ofthe above, it would be advantageous if an apparatus were available which permitted stable and reliable real time measurement of fluorescent indicators of amplification products resulting from any of the available nucleic acid amplification schemes.

~1598~0 Summary of the Invention The invention relates to a system for carrying out real time fluorescence-based measurements of nucleic acid amplification products. In a preferred embodiment of the invention, an excitation beam is focused into a reaction mixture containing (i) a first fluorescent indicator capable of generating a first fluorescent signal whose intensity is proportional to the amount of an amplification product in the volume of the reaction mixture illuminated by the excitation beam and (ii) a second fluorescent indicator homogeneously distributed throughout the reaction mixture and capable of generating a second fluorescent signal proportional to the volume of reaction mixture illuminated by the excitation beam. It is understood that the proportionality of the fluorescent intensities is for a constant set of parameters such as temperature, pH, salt concentration, and the like, that independently influence the fluorescent emissions of organic dyes.
Preferably, the excitation beam is focused into the reaction mixture by a lens through a portion of a wall of a closed reaction chamber containing the reaction mixture. In further preference, the same lens collects the first and second fluorescern signals generated by the first and second fluorescent indicators, respectively, in response to the excitation beam; thus, variability in the collected signal due to misalignment of excitarion and collection optics is avoided. In this embodiment, whenever the lens directs the excitation beam through a portion of a wall of the closed reaction chamber which is not in contact with the reaction mixture, that portion of the wall is heated so that condensation from the reaction mixture does not form in the optical pathway of the fluorescent signals being collected by the leas, thereby removing another source of variability in the collected signal.
In the most preferred embodiment, the reaction chamber is a tube with a closed end, referred to herein as the bottom of the tube, and an open end, referred to herein as the top of the tube, which can be closed with a cap such that a leak-proof seal is formed. In other words, once a reaction mixture is placed in the tube and the cap is attached, a closed reaction chamber is formed. In this most preferred embodiment, (1) the reaction mixture fills a portion of the tube, generally at the bottom of the tube, such that a void is left between the cap of the tube and a top surface of the reaction mixture, (2) the walls of the tube are frosted; that is, they are made of a material that transmits and scatters light, and (3) the lens without contacting the cap focuses the excitation beam through the cap into the reaction mixture through its top surface and collects the resulting fluorescence generated by the first and second fluorescent indicators. As mentioned above, the portion of the tube through which the excitation beam passes-the cap in this embodiment-is heated to prevent the formation of condensation which would introduce an added source of variability in the measurement of the collected fluorescent signals. Potential variability that could arise from sequential analysis of the first and second fluorescent signals is eliminated by simultaneously analyzing the signals by spectrally separating the signal light onto an array of photo detectors, e.g. by di$'racting the signal onto a charged-coupled device (CCD) array.
As discussed more fitlly below, an excitation beam generated by a single light source, e.g. a laser, is conveniently distributed to a plurality of closed reaction chambers by fiber optics. Likewise, the same fiber optics can collect the fluorescent signals from the plurality of reaction chambers for analysis by a single detection and analysis system.
Preferably, the system is employed with the PCR amplification of nucleic acids.
The system of the invemion permits accurate real time monitoring of nucleic amplification reactions by providing apparatus and fluorescent reage~s for generating a stable fluorescent signal proportional to the amount of amplification product and independent of variations in the volume of reaction mixture. The availability of data showing the progress of amplification reactions leads to more accurate estimates of relative starting concemrations of target nucleic acids, to rapid assessment of the efficiency of the amplification reactions, and opens the possibility of reduced reagent usage and feedback reaction control.
Brief Description of the Figures Figure 1 diagrammatically illustrates a preferred embodiment of the sample interface components of the system of the invention.
Figure 2 diagrammatically illustrates a preferred embodimern for simultaneously monitoring a plurality of amplification reactions by sequentially intesogaiing reactions via a fiber optic multiplexer.
Figure 3 shows spectrally separated fluorescent intensity data for a tetramethylrhodamine fluorescent indicator, a fluorescein fluorescent indicator, and instrumern background registered by a CCD array of the preferred embodiment described below.
Figure 4 shows the time dependence of fluorescent signals from a fluorescein dye proportional to the amplification product (first fluorescent indicator) and a tetramethy(rhodamine dye employed as a second fluorescent indicator during a typical PCR

~1~98~~
Figure S shows the cycle dependence of the ratio of the intensities of the fluorescein and tetramethylrhodamine dyes from the same PCR whose time dependent data is shown in Figure 3.
Figure 6 shows data relating the amount of amplification product to cycle number in separate PCRs having different starting concentrations of the same target nucleic acid.
Definitions As used herein, the term "stable" in reference to a fluorescent signal means that the root means square (RMS) deviation in the signal due to noise is less than or equal to two percent of the average signal magnitude. More preferably, stable means that the RMS deviation in the signal due to noise is less than or equal to one percent of the average signal magnitude.
Detailed DescriRtion of the Invention The invention is a fluorescence-based system for monitoring in real time the progress of a nucleic acid amplification reaction. The type of amplification scheme used with the system is not critical, but generally the system requires either the use of a nucleic acid polymecase with exonuclease activity or a population of double stranded DNA which increases during the course of the reaction being monitored.
Exemplary amplification schemes that may be employed with the system of the invention include PCR, ligase-based amplification schemes, such as ligase chain reaction (LCR), Q-beta replicase-based amplification schemes, strand displacement amplification (SDA) schemes, such as described by Walker et al, Nucleic Acids Research, 20: 1691-(1992), and the like. A comprehensive description of nucleic acid amplification schemes is provided by Keller and Manak, DNA Probes, Second Edition (Stockton Press, New York, 1993). Fundamental to the system is the measurement of ratios of fluorescent intensities of a first fluorescent indicator and an internal standard, referred to herein as a second fluorescent indicator. The first and second fluorescent indicators must be specually resolvable. That is, their respective emission spectra must be sufficiently non-overlapping so that separate emission peaks are observed in the combined spectrum. Clearly, the system may be generalized to include a plurality of first fluorescent indicators, e.g. to monitor the simultaneous amplification of several target nucleic acids in a single reaction, so that a plurality of fluorescent intensity ratios are monitored. Several spectrally resolvable dyes suitable for use in such embodiments are disclosed in Fung et al, U.S. patent 4,855,225; Menchen et al, U.S. patent 5,188,934; Bergot et al, International Application PCTlUS90/05565; and like references.

The system includes a sample interface-that is, optical components operationally associated with a closed reaction chamber--which comprises a lens for focusing an excitation beam into the reaction mixture and for collecting the resulting fluorescence and a fiber optic for transmitting both the excitation beam from a light source to the lens and the fluorescent signals from the lens to a detection and analysis means. Preferably, the reaction mixture is contained in a closed reaction chamber to prevent cross-sample contamination, or so-called "carryover." The lens therefore focuses the excitation beam and collects fluorescence through a portion of a wall of the closed reaction chamber. As mentioned above, the preferred reaction chamber is a tube, e.g. having the geometry and volume of a conventional Eppendorf tube.
The tube is closed after the reaction mixture is added by attaching a cap to the open end of the tube. In a preferred embodiment of the sample interface for PCR, the lens directs the excitation beam and collects fluorescence through the cap of the tube, as illustrated in Figure 1. In the illustrated configuration, a first end fiber optic 2 is held by ferrule 4, housing 6, and plate 10 in a co-axial orientation with lens 8. A second end of fiber optic 2 (not shown) is operationally associated with a light source and detection and analysis means, discussed more fitlly below. The distance between the end face of fiber optic 2 and lens 8 is determined by several factors, including the numerical aperture of the fiber optic, the geometry of tube 18, the focal length of lens 8, the diameter of lens 8, and the like. Guidance for selecting values for such variables in any particular embodiment is readily found is standard texts on optical design, e.g. Optics Guide 5 (Melles Griot, Irvine, CA, 1990), or like reference. In the illustrated embodimerrt, lens 8 has a diameter of 8 mm and is composed of material BK7, available from Edmund Scientific (Barrington, Nn. Fiber optic 2 has a numerical aperture of .2. Preferably, the design permits maximal transmission of excitation beam 28 to reaction mixture 22. For example, lens 8, numerical aperture of fiber optic 2, and the distance between the end of fiber optic 2 and lens 8 are selected so that the diameter of lens 8 equals or exceeds the diameter of excitation beam 28 where beam 28 impinges on the lens (as illustrated in Figure 1). Excitation beam 28 is focused through cap 16, void 24, and top surface 26 of reaction mixture 22 to a region approximately 1-3 times the diameter of the fiber optic just below, e.g. 1-3 mm, surface 26. This degree of focusing is not a critical feature of the embodiment; it is a consequence of adapting the sample interface to the geometry and dimensions of a sample holder of a commercially available thermal cycler. In other embodiments, the geometry and dimension may permit a sharper focus into the reaction mixture.
The lens of the invention may have a variety of shapes depending on particular embodiments. For example, the lens may be a sphere, truncated sphere, cylinder, truncated cylinder, oblate spheroid, or truncated oblate spheroid, or the like, and may 2159~3~
be composed of any suitably transparent refractive material, such as disclosed by HIousek, U.S. patent 5,037,199; Hoppe et al, U.S. patent 4,747,87; Moring et al, U.S.
patent 5,239,360; Hirschfield, U.S. patent 4,577,109; or like references.
Fluorescent light generated by excitation beam 28 is collected by lens 8 along approximately the same optical pathway as that defined by excitation beam 28 and focused onto the end of fiber optic 2 for transmission to optical separation and analysis components of the system.
In fitrther preference, the sample interface also includes means for heating the portion of the wall of the reaction chamber used for optical transmission in order to reduce variability due to scatter and/or absorption of the excitation beam and signal from condensation of reaction mixture components. In the embodiment of Figure 1, the portion of the reaction chamber (tube 18) wall used for optical transmission is cap 16. Accordingly, heating element 12 and heat-conductive platen 14 are employed to heat cap 16. Preferably, heating element 12 comprises resistance heating elements and temperature sensors that permit programmed controlled of the temperature of cap 16.
Cap 16 is maintained at a temperature above the condensation points of the components of the reaction mixture. Generally, cap 16 may be maintained at a temperature in the range of 94-110oC. Preferably, cap 16 is maintained at a temperature in the range of about 102oC to about lOSoC since the principal solvent in the reaction mixture is usually water. More preferably, cap 16 is maintained at 103oC.
Preferably, in embodiments employing thermal cycling, the cap-heating componems described above are thermally isolated from heating-conducting componem 20 employed to cyclically control the temperature of reaction mixture 22.
As mentioned above, walls of tube 18 are preferably frosted so that a~
spurious reflections from the walls of heat-conducting component 20 are diffused or scattered to reduce the cotmtbutions such reflections may make to the collected signal.
Walls of ordinarily translucent or transparent tubes are conveniently frosted by etching or roughening.
Selection of appropriate materials for the components described above is well within the skill of an ordinary mechanical engineer. Exemplary criterion for material selection inciude ~) degree ofthermal expansior>, especially for amplification schemes employing thermal cycling, and its affect on the alignment of the optical components, (ii) optical transmission properties in the excitation wavelengths and fluorophore emission wavelengths employed, (iii) chemical inertness of the reaction chamber relative to components of the reaction mixture, (iv) degree to which critical reaction components, e.g. polymerases, target nucleic acids, would tend to adsorb onto chamber walls, (v) minimization of fluorescent materials in the optical pathway, and _7_ the like. Typically, tubes containing ampliYication reaction mixtures are made of polypropylene or like materials.
The sample interface shown in Figure 1 may be employed individually or it may be employed as one of a plurality of identical interfaces in a single instrument, as shown diagrammatically in Figure 2. In the illustrated embodiment, individual sample interfaces 31, arrayed in holder 30 (which may, for example, be a heating block associated with thermal cycler 32, such as described in Mossa et al, European patent application No. 91311090.4, publ. No. 0488769 A2) are connected by fiber optics 34 to fiber optic multiplexer 36, which selectively permits transmission between individual fiber optics and port 35, e.g under user control via a programmed microprocessor. In a preferred configuration, excitation beam 41, generated by light source 52 and controller 54, passes through beam sputter 40 and is focused onto port 35 by lens 38, where it is sequentially directed by fiber optic multiplexer 36 to each of a predetermined set, or subset, of fiber optics 34. Conversely, a fluorescent signal generated in a reaction chambers is collected by lens 8 aad focused onto a fiber optic which, in turn, transmits the signal to a detection and analysis means, possibly via a fiber optic multiplexer. Returning to Figure 2, a fluorescem signal collected by a sample interface is directed to fiber optic multiplexer 36 where it emerges through port 35 and is collected and collimated by lens 38. Lens 38 directs the fluorescent signal to beam sputter 40 which, is turn, selectively directs the signal through cut-off filter 42, which prevents light from the excitation beam from reaching the signal detection components. Beam sputter 40 may be a conventional dichroic mirror, a firlly reflective mirror with an aperture to pass the excitation beam (e.g. as disclosed in U.S.
patent 4,577,109), or like component. After passing through cut-offfilter 42, the fluorescent signal is directed by lens 44 to a spectral analyzer which spectrally separates the fluorescent signal and measures the intensities of a plurality of the spectral components of the signal. Typically, a spectral analyzer comprises means for separating the fluorescent signal into its spectral components, such as a prism, diffraction grating, or the like, and an array of photo-detectors, such as a diode array, a charge-coupled device (CCD) system, an array of bandpass filters and photomultiplier tubes, or the like. In the preferred embodiment ofFigure 2, the spectral analyzer comprises diffraction grating 46 (e.g., model CP-140, Jobin-Yvon, NJ) and CCD array 48 (e.g., model 52135 Princeton Instruments, NJ), which is linked to CCD controller 50.
An exemplary CCD array suitable for analyzing fluorescent signal from fluorescein and tetramethylrhodamine is partitioned into 21 collection bins which span the 500 qm to 650 nm region of the spectrum. Each bin collects light over a 8.5 nm window. Clearly, many alternative configurations may also be employed. An _g_ exemplary application of a CCD array for spectral analysis is described in Karger et al, Nucleic Acids Research, 19: 4955-4962 (1991).
Analyzing the fluorescent signal based on data collected by a spectral analyzer is desirable since components of the signal due to one or more first fluorescent indicators and a second fluorescent indicator (from which intensity ratios are calculated) can be analyzed simultaneously and without the introduction of wavelength-specific system variability that might arise, e.g. by misalignment, in a system based on multiple beam sputters, filters, and photomultiplier tubes.
Also, a spectral analyzer permits the use of "virtual filters" or the programmed manipulation of data generated from the array of photo-detectors, wherein a plurality of discrete wavelength ranges are sampled-in analogy with physical bandpass filters-under programmable control via an associated microprocessor. This capability permits a high degree of flexibility in the selection of dyes as first and second fluorescent indicators.
Generally, the detection and analysis means may be any detection apparatus to provides a readout that reflect the ratio of irnensities of the signals generated by the first and second fluorescent indicators. Such apparatus is well lmow in the art, as exemplified by U.S. patents 4,577,109 and 4,786,886 and references such as The Photonics Design & Applications Handbook, 39th Edition (Laurin Publishing Co., Pittsfield, MA, 1993).
Preferably, the system of the invention is employed to monitor PCRs, although it may also be employed with a variety of other amplification schemes, such as LCR.
Descriptions of and guidance for conducting PCRs is provided in an extensive literature on the subject, e.g. including Innis et al (cited above) and McPherson et al (cited above). Briefly, in a PCR two oligonucleotides are used as primers for a series of synthetic reactions that are catalyzed by a DNA polymerise. These oligonucleotides typically have different sequences and are complementary to sequences that (i) lie on opposite strands of the template, or target, DNA and (ii) flank the segment of DNA that is to be amplified. The target DNA is first denatured by heating in the presence of a large molar excess of each of the two oligonucleotides and the four deoxynucleoside triphosphates (dNTPs). The reaction mixture is then cooled to a temperature that allows the oligonucleotide primers to anneal to their target sequences, after which the annealed primers are extended with DNA polymerise.
The cycle of denaturation, annealing, and extension is then repeated many times, typically 25-35 times. Because the products of one round of amplification serve as target nucleic acids for the next, each successive cycle essentially doubles the amount of target DNA, or amplification product.
As mentioned above an important aspect of the invention is the fluorescent dyes used as the first and second fluorescent indicators. By examining the ratio of the fluorescent intensities of the indicators, the effects of most sources of systematic variability, which would be apparent in the intensities alone, are eliminated.
Generally, in accordance with the invention, the first fluorescent indicator may be a complex-forming dye or a dye covalently attached to an oligonucieotide probe which is degraded during polymerization steps to generate a signal. This later embodiment relates to the so-called "Tacman" approach, described by Holland et al, Proc.
Natl.
Acad. Sci., 88: 7276-7280 (1991). As used herein, the term "complex-forming"
in reference to a dye means that a dye is capable of forming a stable non-covalent complex with either double stranded or triple stranded nucleic acid structures, usually DNA, and that the dye's fluorescent characteristics are substantially different in the complexed state as compared to a non-complexed, i.e. usually free-solution, state.
Preferably, the quantum efficiency of fluorescence of an complex-forming dye is enhanced in the complexed state as compared to the free-solution state, thereby resulting in enhanced fluorescem upon complex formation. Exemplary complex-forming dyes include et6idium bromide, propidium iodide, thiazole orange, acridine orange, daunomycin, mepacrine, 4',6'-diaminidino-2-phenylindole (DAPn, oxazole orange, bisbenzitnidaxole dyes, such as Hoechst 33258 and Hoechst 33342, and heterodimers of various intercalating dyes, such as ethidium, acridine, thiazolium, and oxazolium dyes (Imown by their acronyms POPRO, BOPRO, YOPRO, and TOPRO), and like dyes, which are descn'bed in the following references: Haugland, pgs.

229 in Handbook of Fluorescent Probes and Research Chemicals, 5th Edition (Molecular Probes, Inc., Eugene, 1992); Glazer et al, Proc. Natl. Acad. Sci., 87: 3851-3855 (1990); Srinivasan et al, Applied and Theoretical Electrophoresis, 3: 235-(1993); Kapuscinski et al, Anal. Biochem., 83: 252-257 (1977); Fall, Anal.
Biochem., 70: 635-638 (1976); Setaro et al, Anal. Biochem, 71: 313-317 (1976); and Latt et al, J. Iqistochem. Cytochem., 24:24-33 (1976); and Rye et al, Nucleic Acids Research, 20:
2803-2812 (1992). Preferably, when complex-forming dyes are employed as first fluorescent indicators, such dyes are selected from the group consisting of t6iazole orange, ethidium bromide, and TOPRO.
Dyes employed as second fluorescent indicators include fluorescent dyes whose fluorescent characteristics are substantially unaffected by the presence or association with nucleic acids, particularly double stranded DNA. Such dyes may include virtually any fluorescent dye fulfilling this criterion which is also spectrally resolvable from whatever first fluorescent indicators that are employed. Preferred second fluorescent indicators include rhodamine dyes and fluorescein dyes. More preferably, the second fluorescegt indicator is tetramethylrhodamine or 2',4',5',T,-tetrachloro-4,7-dichlorofluorescein, the latter being disclosed by Menchen et al, U.S. patent 5,188,934.

In a preferred embodiment, a first fluorescent indicator and a second fluorescent indicator are both covalently attached to an oligonucleotide probe as described by Lee et al, Nucleic Acid Research, 21: 3761-3766 (1993). More specifically, fluorescein is used as the first fluorescent indicator and tetramethylrhodamine is used as the second fluorescent indicator such that the tetramethylrhodamine moiety substantially quenches any fluorescent emissions by the fluorescein moiety. Thus, when both dyes are attached to the same oligonucleotide, only the tetramethylrhodamine is capable of generating a fluorescent signal.
When the oligonucleotide is cleaved, e.g. via the 5'->3' exonuclease activity of a DNA
polymerise, separating the two dyes, the fluorescein became capable of generating a fluorescent signal. Preferably, in this embodiment, the excitation beam is generated from the 488 am emission line of an argon ion laser. In accordance with the invention, in a PCR the production of "free" fluorescein in this embodiment is proportional to the amount of DNA synthesis catalyzed by the DNA polymerise employed, and hence, the amount of amplification product. In this embodiment, preferably the first fluorescent indicator is fluorescein, e.g. 6-FAM (available from Applied Biosystems, Foster City), and the second fluorescent indicator is either tetramethylrhodamine or 2',4',5',T;
tetrachloro~,7-dichlorofluorescein.
Such oligonucleotide probes of the invention can be synthesized by a number of approaches, e.g. Ozaki et al, Nucleic Acids Research, 20: 5205-5214 (1992);
Agrawal et al, Nucleic Acids Research, 18: 5419-5423 (1990); or the like. Preferably, the oligonucleotide probes are synthesized on an automated solid phase DNA
synthesizer using phosphoramidite chemistry, e.g. Applied Biosystems, Inc. model 392 or synthesizer (Foster City, CA). The first and second fluorescent indicators can be covalently attached to predetermined nucleotide of an oligonucleotide by using nucleoside phosphoramidite monomers containing reactive groups. For example, such reactive groups can be on a phosphate, or phosphate analog, e.g. Agrawal et al (cited above), on the 5' hydroxyl when attachment is to the 5' terminal nucleotide, e.g. Fung et al, U.S.
patent 4,757,141 or Hobbs Jr., U.S. patent 4,997,928, and on base moieties, e.g. as disclosed by Ruth U.S. patent 4,948,882; Haralambidis et at, Nucleic Acids Research, 15:
4857-4876 (1987); Urdea et al, U.S. patent 5,093,232; Cruickshank U.S. patent 5,091,519; Hobbs Jr. et al, U.S. patent 5,151,507; or the like. Most preferably, nucleotides having pyrimidine moieties are derivatized. In further preference, the 3' terminal nucleotide of the oligonucleotide probe is blocked or rendered incapable of extension by a nucleic acid polymerise. Such blocking is conveniently carried out by the attachmept of a phosphate group, e.g. via reagents described by Horn and Urdea, Tetrahedron Lett., 27: 4705 (1986), and commercially available as 5' Phosphate-ONtM
from Clontech Laboratories (Palo Alto, California). Preferably, the oligonucleotide probe is in the range of IS-60 nucleotides in length. More preferably, the oGgonucleotide probe is in the range of 18-30 nucleotides in length.
The separation of the first and second fluorescent indicators within the oligonucleotide probe can vary depending on the nature of the first fluorescent indicator and second fluorescent indicator, the manner in which they are attached, the illumination source, and the like. Guidance concerning the selection of an appropriate distance for a given embodiment is found in numerous references on resonant energy transfer between fluorescent molecules and quenching molecules (also sometimes referred to as "donor"
molecules and "acceptor" molecules, respectively), e.g. Stryer and Haugland, Proc. Natl.
Acad. Sci., 58: 719-726 (1967); Clegg, Meth. Enzymol., 211: 353-388 (1992);
Cardullo et al, Proc. Natl. Acad. Sci., 85: 8790.8794 (1988); Ozaki et al (cited above);
Haugland (cited above); Heller et a(, Fed. Proc., 46: 1968 (1987); and the like. The first and second fluorescent indicators must be close enough so that substantially all, e.g.
90%, of the fluorescence from the first fluorescent indicator is quenched. Typically, for energy transfer-based quenching, the distance between the first and second fluorescern indicators should be within the range of 10-100 angstroms. 1n one embodiment, the first and second fluorescent indicators are separated by between about 4 to 10 nucleotides, and more preferably, they are separated by between 4 and 6 nucleotides, with the proviso that there are no intervening secondary structures, such as hairpins, or the like.
Preferably, either the first or second fluorescent indicator is attached to the 5' terminal nucleotide of the oligonucieotide probe.
In another embodiment, an oligonucieotide probe is provided with first and second fluorescent indicators attached at opposite ends. This configuration permits a more conveniently synthesized probe that in free solution forms a random coil bringing the first and second fluorescent indicators within energy transfer range. Thus, in the non-hybridized state, the excited fluorescent moiety is quenched. In the hybridized state the fluorescent moiety and quencher are drawn apart so that energy transfer become negligible permitting the excited fluorescent moiety to emit fluorescence.
There are many linking moieties and methodologies for attaching indicator molecules to the 5' or 3' termini of oligonucleotides, as exemplified by the following references: Eckstein, editor, Oligonucleotides and Analogues: A
Practical Approach ()ItL Press, Oxford, 1991); Zuckerman et al, Nucleic Acids Research, 15: 5305-5321 (1987)(3' thiol group on oligonucleotide); Sharma et al, Nucleic Acids Research, 19: 3019 (1991)(3' su(thydryl); Giusti et al, PCR
Methods and Applications, 2: 223-227 (1993) and Fung et a(, U.S. patent 4,757,141 (5' phosphoamino group via AminolinkTh't II available from Applied Biosystems, Foster City, CA); Stabinsky, U.S. patent 4,739,044 (3' aminoalkylphosphoryi group); Agrawal et al, Tetrahedron Letters, 31: 1543-1546 (1990)(attachment via phosphoramidate linkages); Sproat et al, Nucleic Acids Research, 15: 4837 (1987)(5' mercapto group); Nelson et al, Nucleic Acids Research, 17: 7187-7194 (1989)(3' amino group); and the like.
Preferably, commercially available linking moieties are employed that can be attached to an oligonucleotide during synthesis, e.g. available from Clontech Laboratories (Palo Alto, CA).
Rhodamine and fluoresceia dyes are also conveniently attached to the 5' hydroxyl of as oligonucleotide at the conclusion of solid phase synthesis by way.of dyes derivatized with a phosphoramidite moiety, e.g. Woo et al, U.S. patent 5,231,191; gad Hobbs, Jr. U.S. patern 4,997,928.
Clearly, related embodiments of the above may be employed wherein the first fluorescent indicator is attached to as oligonucleotide probe with another non-fluorescent quenching molecule, instead of a second fluorescent indicator. In such embodiments, the second fluorescent indicator could be virtually a~ spectrally resolvable fluorescent dye that did not interact with the amplification products.
EXPERIMENTAL
Real time monitoring of PCR am~olification of DNA encoding B-actin from various starting concentrations oftar~NA
A 296 basepair segment of a target DNA encoding human ~3-actin was amplified by PCR from various starting amourns in the range of 5x103 to 1x106 copies of target DNA. The following primers gad probe were employed:
5'-TCACCCACACTGTGCCCATCTACGA
(forward primer SEQ. LD. No.: 1) 5'-CAGCGGAACCGCTCATTGCCAATGGT
(reverse primer SEQ. LD. No.: 2) 5'-A(FAI~TGCCCT('TMR)CCCCCATGCCATCCTGCGT
(probe SEQ. LD. No.: 3) wherein "FAM" indicates a fluorescein molecule coupled to the oligonucleotide by reacting an NHS-ester group attached to the fluorescein's 6 carbon with a 5'-aminophosphate attached to the 5'-terminal deoxyadenosine of the oligonucleotide in ~._ accordance with Fung et al, U.S. patent 5,212,304; and wherein "TMR" indicates a tetramethylahodamine molecule coupled to the base moiety of the adjacent thymidine via the amino linking agent disclosed by Urdea et al, U.S. patent 5,093,232.
PCRs were carried out in 0.2 mL MicroAmp tubes (Perkin-Elmer, Norwallc, CT) with the following components: 10 mM Tris-HCI, pH 8.3, 50 mM KCI, 3.5 mM
MgCl2, 200 EtM each of the nucleoside triphosphates (with dUTP substituted for dTTP
in accordance with U.S. patent 5,035,996 to prevent carryover contamination), 300 nM
each of forward and reverse primers, AmpliTaq (Perkia-Eliner, Norwalk, CT) at 0.05 U/p.L. To this mixture was added 5 EtL Raji DNA (Applied Biosystems, Foster City, CA) at 10 ng/~L, 5 itI. probe at 2 pM, and 1 EtL uracil N glycosylase at 1 unit/p.L to bring the reaction volume to 51 ltl,. Heating and cooling cycles were carried out in a model 9600 Thermal Cycler (Peridn-Elmer, Norwallc, CT) fitted with a sample holder cover containing the sample interface components of the invention. The following temperature profile was employed: hold for 2 minutes at SOoC; hold for 10 mimrtes at 95oC; cycle through the following temperatures 40 times: 92oC for 15 seconds, 54oC
for 15 seconds, 72oC for 1 minute; then hold at 72oC.
1 figure 3 illustrates data showing the emission specria of the fluorescein and tetramethylrhodamine dyes employed as indicators above and fluorescence due to extraneous sources in the system.
Figure 4 illusuates data showing fluorescein fluorescent imensity and tetramethylrhodamine fluorescent intensity as a fimction of cycle number. The high frequency oscillations in i~ensity reflect the temperature dependence of the fluorescent emission of the two dyes. An increase in base line fluorescence for both dyes beiweea cycles 10 and 28 is a system-based variation. In Figure 5, which illustrates the ratio of fluorescein-to-tetramethyliitodamine fluorescent intensity from the same data, the system-based variation is eliminated and the RMS of fluctuations in the readout signal, that is, the ratio of fluorescent intensities, is less than 1% of the average magnitude of the measured ratio.
Figure 6 illustrates data from PCR of the (i-actin DNA starting from amounts ranging from 5000 target molecules.to 106 target molecules as indicated in the figure.
* Trademarks SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Timothy M. Woudenberg, Kevin S. Bodner, Charles R. Connell, Alan M. Gan2, Lincoln J. McBride, Paul G. Saviano, John Shigeura, David H. Tracy, Eugene F. Young (ii) TITLE OF INVENTION: System for real time detection of nucleic acid amplification products (iii) NUMBER OF SEQUENCES:
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Stephen C. Macevicz, Applied Biosystems, Inc.
B STREET: 850 Lincoln Centre Drive C CITY: Foster City D STATE: California E COUNTRY: USA
F ZIP: 94404 (v) COMPUTER READABLE FORM:
A) MEDIUM TYPE- 3.5 inch diskette B) COMPUTER: IBM compatible C OPERATING SYSTEM: Windows 3.1/DOS 5.0 ~D; SOFTWARE: Microsoft Word for Windows, vets. 2.0 (vi) CURRENT APPLICATION DATA:
A APPLICATION NUMBER:
B FILING DATE:
~C~ CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
A APPLICATION NUMBER: 08/235,411 ~B; FILING DATE: 29-APR-94 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Stephen C Macevicz ((B) REGISTRATION NUMBER- 30,285 C) REFERENCE/DOCKET NUMBER: 4241wo (ix) TELECOMMUNICATION INFORMATION:
A) TELEPHONE: (415) 358-7855 ~B) TELEFAX: (415) 358-7794 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
~~ LENGTH: 25 nucleotides TYPE: nucleic acid C STRANDEDNESS: single D TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 26 nucleotides ~B~ TYPE: nucleic acid ((C))) STRANDEDNESS: single D TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
A) LENGTH: 26 nucleotides ~B~ TYPE: nucleic acid ((C)) STRANDEDNESS: single D TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An apparatus for monitoring the formation of a nucleic acid amplification reaction product in real time, the apparatus comprising:
a sample holder for holding a sample of nucleic acids to be amplified;
a fiber optic cable for illuminating a volume of the sample with an excitation beam;
a lens co-axially disposed with the fiber optic cable for focusing the excitation beam into the volume of the sample and for collecting from the sample and transmitting to the fiber optic cable a first fluorescent signal whose intensity is proportional to the concentration of the amplification reaction product and a second fluorescent signal whose intensity is proportional to the volume of the sample illuminated by the excitation beam; and a detection and analysis mechanism for receiving the first and second fluorescent signals from the fiber optic cable at a plurality of times during a nucleic acid amplification, the detection and analysis mechanism measuring the intensities of the first and second fluorescent signals at the plurality of times and producing a plurality of corrected intensity signals, each corrected intensity signal corresponding to a relationship between the intensities of the first and second fluorescent signals at a given time.

2. The apparatus according to claim 1 wherein the detection and analysis mechanism provides a readout corresponding to the plurality of corrected intensity signals as a function of time.

3. The apparatus according to claim 1 wherein the apparatus includes:
a plurality of sample holders for holding a plurality of samples;
a plurality of fiber optic cables for illuminating volumes of the plurality of samples;
a plurality of lenses, each co-axially disposed with a first end of a fiber optic cable for focusing an excitation beam into a sample; and a fiber optic multiplexer which couples the detection and analysis mechanism to a second end of each of the plurality of fiber optic cables.

4. The apparatus according to claim 1 wherein the sample holder includes a removable reaction chamber for holding the sample.

5. The apparatus according to claim 1 wherein the removable reaction chamber is sealable.

6. The apparatus according to claim 1 wherein the sample holder includes a sealable reaction chamber for holding the sample.

7. The apparatus according to claim 1 wherein the sample holder includes an optical interface through which the excitation beam is transmitted from the lens into the sample.

8. The apparatus according to claim 7 wherein the sample holder includes a sealable reaction chamber for holding the sample, the optical interface forming a wall of the reaction chamber.

9. The apparatus according to claim 7 wherein the apparatus further includes a mechanism for heating the optical interface to reduce condensation of the sample on the optical interface.

10. The apparatus according to claim 9 herein the sample holder includes a sealable reaction chamber for holding the sample, the optical interface forming a wall of the reaction chamber.

11. The apparatus according to claim 7 wherein the sample holder includes a removable reaction chamber for holding the sample, the optical interface forming a wall of the reaction chamber which covers at least a portion of the sample and which is separated from the sample by an air gap.

12. The apparatus according to claim 1 wherein the nucleic acid amplification includes a plurality of amplification cycles and the detection and analysis mechanism receives the first and second fluorescent signals from the fiber optic cable at least once per amplification cycle and measures the intensities of the first and second fluorescent signals at least once per amplification cycle.

13. The apparatus according to claim 12 wherein the detection and analysis mechanism produces at least one corrected intensity signal per amplification cycle.

14. A method for monitoring the formation of a nucleic acid amplification reaction product in real time comprising:
taking a sample holder containing a nucleic acid sequence to be amplified to form a nucleic acid amplification reaction product, a first fluorescent indicator which produces a first fluorescent signal when illuminated by an excitation beam whose intensity is proportional to a concentration of the amplification reaction product in the sample, and a second fluorescent indicator which produces a second fluorescent signal when illuminated by the excitation beam whose intensity is proportional to a volume of sample illuminated by the excitation beam;
performing an amplification of the nucleic acid sequence in the sample holder;
transmitting an excitation beam into the sample holder at a plurality of times during the amplification and measuring the intensities of the first and second fluorescent signals at the plurality of times; and monitoring the formation of the nucleic acid amplification reaction product in real time by calculating a plurality of corrected intensity signals, each corrected intensity signal corresponding to a relationship between the intensity of the first and second fluorescent signals measured at the plurality of times, a change in the corrected intensity signal over time indicating the formation of the nucleic acid amplification reaction product.

15. A method for monitoring the formation of multiple nucleic acid amplification reaction products in real time comprising:
taking multiple sample holders, each sample holder containing a nucleic acid sequence to be amplified to form a nucleic acid amplification reaction product, a first fluorescent indicator which produces a first fluorescent signal when illuminated by an excitation beam whose intensity is proportional to a concentration of the amplification reaction product in the sample, and a second fluorescent indicator which produces a second fluorescent signal when illuminated by the excitation beam whose intensity is proportional to a volume of sample illuminated by the excitation beam;
performing an amplification of the nucleic acid sequences in the multiple sample holders;
transmitting an excitation beam into the multiple sample holders at a plurality of times during the amplification and measuring the intensities of the first and second fluorescent signals at the plurality of times; and monitoring the formation of nucleic acid amplification reaction products in the multiple sample holders in real time by calculating a plurality of corrected intensity signals, each corrected intensity signal corresponding to a relationship between the intensity of the first and second fluorescent signals measured at the plurality of times, a change in the corrected intensity signal over time indicating the formation of the nucleic acid amplification reaction product.

16. A method for monitoring the formation of multiple nucleic acid amplification reaction products in real time comprising:
taking a sample holder containing a plurality of nucleic acid sequences to be amplified to form a plurality of nucleic acid amplification reaction products, a plurality of first fluorescent indicators which produce a first fluorescent signal when illuminated by an excitation beam whose intensity is proportional to a concentration of the amplification reaction product in the sample, and a second fluorescent indicator which produces a second fluorescent signal when illuminated by the excitation beam whose intensity is proportional to a volume of sample illuminated by the excitation beam;
performing an amplification of the nucleic acid sequence in the sample holder;
transmitting an excitation beam into the sample holder at a plurality of times during the amplification and measuring the intensities of the first and second fluorescent signals at the plurality of times; and monitoring the formation of the plurality of nucleic acid amplification reaction products in the sample holder in real time by calculating a plurality of corrected intensity signals for each of the plurality of nucleic acid sequences in the sample holder at the plurality of times, each corrected intensity signal corresponding to a relationship between the intensity of the first and second fluorescent signals measured at the plurality of times, a change in the corrected intensity signal over time indicating the formation of the nucleic acid amplification reaction product.

17. A method for monitoring the formation of a nucleic acid amplification reaction product in real time comprising:
taking a sample holder containing a nucleic acid sequence to be amplified to form a nucleic acid amplification reaction product, and first and second fluorescent indicators covalently attached to an oligonucleotide capable of hybridizing to the amplification reaction product, the first fluorescent indicator producing a first fluorescent signal when illuminated by the excitation beam whose intensity is proportional to the concentration of amplification reaction product in the sample, the second fluorescent indicator producing a second fluorescent signal when illuminated by the excitation beam whose intensity is proportional to the volume of the sample illuminated by the excitation beam, the second fluorescent indicator also quenching the fluorescence of the first fluorescent indicator;
performing an amplification of the nucleic acid sequence in the sample holder;
transmitting an excitation beam into the sample holder at a plurality of times during the amplification and measuring the intensities of the first and second fluorescent signals at the plurality of times; and monitoring the formation of the nucleic acid amplification reaction product in real time by calculating a plurality of corrected intensity signals, each corrected intensity signal corresponding to a relationship between the intensity of the first and second fluorescent signals at the plurality of times, a change in the corrected intensity signal over time indicating the formation of the nucleic acid amplification reaction product.

18. The method according to any one of claims 14-17 wherein the first fluorescent indicator is a complex-forming dye.

19. The method according to any one of claims 14-17, further including the step of sealing the sample within the sample holder prior to transmitting an excitation beam into the sample holder.

20. The method according to any one of claims 14-17 wherein the sample holder includes an optical interface through which the excitation beam is transmitted to the sample, the sample holder also including an air gap separating the optical interface from the sample, the method further including the step of heating the optical interface to reduce condensation of the sample on the optical interface.

21. The method according to claim 20, further including the step of sealing the sample within the sample holder prior to transmitting an excitation beam into the sample.

22. The method according to any one of claims 14-17 wherein the step of taking a sample holder includes:
adding a sample to a reaction chamber which is removable from the sample holder; and adding the removable reaction chamber to the sample holder.

23. The method according to claim 22, further including the step of sealing the sample within the removable reaction chamber.

24. The method according to claim 23 wherein the removable reaction chamber includes an optical interface through which the excitation beam is transmitted from the lens to the sample and an air gap separating the optical interface from the sample, the method further including the step of heating the optical interface to reduce condensation of the sample on the optical interface.

25. The method according to any one of claims 14-17 wherein performing the amplification includes performing at least one cycle of a polymerase chain reaction.

26. The method according to any one of claims 14-17 wherein performing the amplification includes performing at least one cycle of a ligase chain reaction.

27. The method according to any one of claims 14-17 wherein performing the amplification includes performing a plurality of cycles of an amplification reaction; and transmitting an excitation beam into the sample holder at a plurality of times during the amplification includes transmitting the excitation beam at least once per amplification cycle.

28. The method according to claim 27 wherein monitoring the formation of the nucleic acid amplification reaction product includes calculating at least one corrected intensity signal per amplification cycle.

29. An apparatus comprising:
a sample holder for holding a reaction chamber which includes an optical interface;
a fiber optic cable for delivering an excitation beam to a sample housed within the reaction chamber and for receiving light emitted by the sample; and a lens co-axially disposed with the fiber optic cable and positioned outside the reaction chamber for focusing the excitation beam through the optical interface and within a volume of the sample and for collecting and transmitting to the fiber optic cable light emitted within the volume of the sample.

30. An apparatus comprising:
a reaction chamber which includes an optical interface;
a fiber optic cable for delivering an excitation beam to a sample housed within the reaction chamber and for receiving light emitted by the sample; and a lens co-axially disposed with the fiber optic cable and positioned outside the reaction chamber for focusing the excitation beam through the optical interface and within a volume of the sample and for collecting and transmitting to the fiber optic cable light emitted within the volume of the sample.

31. The apparatus according to claim 29 wherein the sample holder includes a removable reaction chamber for holding the sample.

32. The apparatus according to claim 30 wherein the reaction chamber is removable.

33. The apparatus according to any one of claims 29-32 wherein the optical interface is a wall of the reaction chamber.

34. The apparatus according to any one of claims 29-33 wherein the lens is not in contact with the reaction chamber.

35. The apparatus according to any one of claims 29-33 wherein the lens is separated from the reaction chamber by an air gap.

36. The apparatus according to any one of claims 29-33 wherein the lens focuses the excitation beam through an air gap positioned between the optical interface and the sample.

37. The apparatus according to any one of claims 29-33 wherein the apparatus further includes a mechanism for heating the optical interface to reduce condensation of the sample on the optical interface.

38. The apparatus according to any one of claims 29-33 wherein the apparatus further includes a detection and analysis mechanism for receiving first and second fluorescent signals from the fiber optic cable.

39. The apparatus according to claim 29 wherein the apparatus includes:
a plurality of sample holders for holding a plurality of reaction chambers which each include an optical interface;
a plurality of fiber optic cables for delivering an excitation beam to samples housed within the plurality of reaction chambers and for receiving light emitted by the samples; and a plurality of lenses co-axially disposed with the fiber optic cables and positioned outside the reaction chambers for focusing the excitation beams through the optical interfaces and within volumes of the samples and for collecting and transmitting to the fiber optic cables light emitted within the volumes of the samples.

40. The apparatus according to claim 30 wherein the apparatus includes:
a plurality of reaction chambers which each include an optical interface;
a plurality of fiber optic cables for delivering an excitation beam to samples housed within the plurality of reaction chambers and for receiving light emitted by the samples; and a plurality of lenses co-axially disposed with the fiber optic cables and positioned outside the reaction chambers for focusing the excitation beams through the optical interfaces and within volumes of the samples and for collecting and transmitting to the fiber optic cables light emitted within the volumes of the samples.

41. A method for monitoring the formation of a nucleic acid amplification reaction product comprising:
amplifying a nucleic acid sequence in a sample in a reaction chamber which includes an optical interface;
focusing an excitation beam within a volume of the sample using a lens positioned outside the reaction chamber;
and collecting light emitted within the volume of the sample using the lens.

42. The method according to claim 41 wherein the lens is not in contact with the reaction chamber.

43. The method according to claim 41 wherein the lens is separated from the reaction chamber by an air gap.

44. The method according to claim 41 wherein the lens focuses the excitation beam through an air gap positioned between the optical interface and the sample.

45. The method according to claim 41 wherein the sample is sealed within the reaction chamber, the method further including the step of heating the optical interface to reduce condensation of the sample on the optical interface.

46. The method according to claim 41, further including the step of transferring the collected emitted light to a detection and analysis mechanism.